The invention relates to the fields of drug discovery and nuclear magnetic resonance spectroscopy.
Nucleic acids (DNA and RNA) are biopolymers in which each monomer contains a negatively charged phosphate ester group. This large negative charge is balanced by interaction of the nucleic acid with small cationic species, including protonated amines, and monovalent and divalent metal ions. In addition to non-specific electrostatic interactions, structured nucleic acids have specific metal ion binding sites. Examples of specific divalent metal ion binding by RNA molecules can be seen in the group III-V intron of the tetrahymina ribozyme, ribosomal RNA, and RNA aptamers. Such interactions may be mediated by direct coordination of the bases or phosphates to the metal ion, or by hydrogen bonding between water molecules coordinated to the metal ion and the nucleotide bases.
Divalent metal ions can be bound by either specific interactions with discrete metal binding sites or non-specifically via coulombic interaction. For non-specific interactions, binding is described by the polyelectrolyte condensation model. Binding affinities are dependant on the ionic strength of the solution and the nature of the cationic form of the supporting electrolyte. This dependence on the nature and concentration of the supporting electrolyte is to be expected since the divalent ion will have to compete with the electrolyte cation for both specific and non-specific interactions. Non-specific affinities can be represented by interactions of Mg2+ with poly-uridine (poly-U). In 10 mM NaCl, 96 μM Mg2+, the apparent association constant for poly-U/Mg binding is reported as 1.86×103 M−1nt−1, where nt is the number of nucleotides in the strand. When other bases are incorporated into the RNA strand, binding of the metal to the bases is possible. Under similar conditions to those of the poly-U study, poly-A has a reported Mg2+ binding affinity of 10.7×103 M−1nt−1, a 5.7 fold increase over poly-U. This difference must be attributed to contributions from base/metal interactions, since the two nucleic acids share the same charge distribution along their backbones. Specific metal binding sites having affinities in the micromolar range have been reported for transfer RNAs and the hammerhead ribozyme. Up to 8 Mn2+ ions bind to the Hammerhead ribozyme with KD values ranging from 4 to 500 μM.
Since divalent metal ions are necessary structural elements of some molecules of RNA and since the RNA molecules exchange bound ions with ions free in the bulk solution, sites for binding metal ions are natural candidates for drug targets. Aminoglycoside antibiotics are believed to act by displacing divalent metal ions from specific binding sites on RNA. Aminoglycosides are reported to displace Mg2+ from specific sites on a variety of RNA molecules including a model of the ribosomal A, the hammerhead ribozyme, the tetrahymina group I intron, and RNase P.
The magnetic relaxation properties of NMR active nuclei can be very sensitive to paramagnetic metal ions. Thermodynamic and structural aspects of macromolecules that bind metal ions have been studied by exploiting this effect. In the majority of works, the effects of the paramagnetic metal ion on the magnetic resonances of 1H of the biological molecule have been determined in order to study the geometry of the metal binding site. In addition, there have been a more limited number of studies where the metal binding sites have been examined using external small species such as water or fluoride ion.
Paramagnetic relaxation enhancement of the magnetic resonance of 1H nuclei of solvent water has been used to study metal binding by a variety of biological macromolecules qualitatively. This approach has been used to characterize the binding of Mn2+ to transfer RNA and ribosomal RNA among other molecules. Another approach for paramagnetic relaxation enhancement that has seen limited use is the use of 19F fluoride as a probe. Relaxation enhancements of this resonance by superoxide dismutases have been reported. Metal ion binding studies where the perturbations observed are of the macromolecular 1H resonances have also been reported. Molecules studied include ribozymes and smaller RNA fragments. Water 1H resonances, however are not very sensitive to paramagnetic relaxation, and 19F measurements require 19F NMR.
There exists a need for sensitive methods for measuring the binding and inhibition of binding of species to nucleic acids. These methods may find use in the field of drug discovery.